Alcohol consumption produces a variety of pathological effects, including the fetal alcohol syndrome, liver and brain damage, and an increased risk of certain types of cancers. The association between ethanol consumption and cancer indicates that alcohol intake results in effects on genomic DNA. Mechanisms by which ethanol can produce DNA damage are: 1) direct adduction of DNA by acetaldehyde, the major metabolite of ethanol; 2) the generation of DNA damaging oxygen radicals via by cytochrome p4502E1 (CYP2E1), which is induced by ethanol in liver and brain. Our hypothesis is that genotoxicity occurs when the level of ethanol-induced DNA damage overwhelms the capacity of the relevant DNA repair systems. Thus the level of DNA repair activity in cells is a crucial determinant of alcohol-induced DNA toxicity. Ongoing work focuses on a detailed understanding of DNA repair mechanisms in target tissues for ethanol toxicity, in particular, the brain. We have developed the first in vitro assay for the nucleotide excision repair (NER) pathway in adult brain tissue. NER repairs some types of oxygen radical damage to DNA, and is critical for protecting neurons against endogenous oxidative DNA damage. Induction of CYP2E1 by ethanol would also result in increased levels of reactive oxygen species and oxidative DNA damage. This system is being used to better characterize the NER pathway in brain cells. To better understand the role of different DNA repair pathways in addressing ethanol-induced DNA damage, we are examining the effects of ethanol, acetaldehyde, and elevated levels of CYP2E1 on cellular toxicity in cell lines and whole animals lacking specific DNA repair pathways. These experiments will determine the relative role of the different DNA repair pathways in protecting cells against different types of ethanol-induced DNA damage. This work may have important implications for human beings with deficiencies in these pathways. Another major focus is on N2-ethyl deoxyguanosine, the major DNA adduct produced by acetaldehyde. This adduct is undetectable in normal liver but accumulates in the DNA of mice fed alcohol. We are assessing whether the adduct is a substrate for DNA repair, and what type of repair is involved. We are also assessing the mutagenicity of this adduct in mammalian cells.